|
|
Construction and Biological Characteristics Analysis of crp Gene Deletion Strain of Haemophilus parasuis Type 4 |
XU Yin-Di*, WANG Zhi-Fang, JIAO Wen-Qiang, ZHU Wen-Hao, LI Hai-Li, WANG Ke-Ling |
Institute of Animal Husbandry and Veterinary Research, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China |
|
|
Abstract Haemophilus parasuis (HPS) is the pathogen causing Glasser's disease, which is one of the most serious bacterial pathogens that harm the health of pigs clinically. There are many serotypes of HPS, serotype 4 is the most isolated serotype and the more serious serotype. crp gene encodes cAMP receptor protein (crp) and crp is one of the most important systemic regulatory factors and plays a crucial role in adapting to environmental changes during bacterial infection. In order to study the effect of crp gene on biological characteristics of HPS type 4 (HPS4) such as growth, resistance and virulence, the crp gene deletion strain Δcrp of HPS type 4 clinical isolate HPS4 was constructed by natural transformation method. Morphological characteristics, growth characteristics, biofilm formation, tolerance pressure, serum bactericidal activity, iron utilization and mice (Mus musculus) infection assays of HPS4 and Δcrp were studied. The results showed that crp deletion strain Δcrp of clinical isolate of HPS4 was successfully constructed in this study. After crp was deleted in HPS4, the growth was significantly slowed down, the biofilm formation ability was significantly weakened, the tolerance to osmotic pressure, oxidative stress and heat stress was decreased, the serum survival ability was decreased, the iron utilization was decreased and the virulence of mice was decreased. The results indicated that crp gene had significant effects on growth, resistance and virulence of HPS4. This study provides basic data for further exploring the function of crp gene and screening attenuated strains of HPS4.
|
Received: 14 September 2022
|
|
Corresponding Authors:
* 445177674@qq.com
|
|
|
|
[1] 王治方, 徐引弟, 朱文豪, 等. 2019. 副猪嗜血杆菌流行优势菌株调查和耐药性分析 [J]. 中国兽医学报, 39(10):1942-46. (Wang Z F, Xu Y D, Zhu W H, et al. 2019. In-vestigation of prevailing strains and drug resistance anal-ysis of Haemophilus parasuis[J]. Chinese Journal of Vet-erinary Science, 39(10): 1942-1946.) [2] 徐引弟, 鲁杨超, 王治方, 等. 2018. 4 型副猪嗜血杆菌的分离鉴定与生物学特性研究 [J]. 黑龙江畜牧兽医, 13:131-134, 246. (Xu Y D, Lu Y C, Wang Z F, et al. 2018. Isolation, identification and biological characteristics of Haemophilus parasuis type 4[J].Heilongjiang Animal Sci-ence and Veterinary Medicine, 13: 131-134, 246.) [3] 闫雪锋, 肖五淀, 古从伟, 等. 2021. 副猪嗜血杆菌 ptsG 基因缺失株的构建及其部分生物学特性分析[J]. 农业生物技术学报, 29(12): 2387-95. (Yan X F, Xiao W D, Gu C W, et al. 2021. Construction of Glaesserella parasuis ptsG mutants strain and analysis of its partial biological char-acteristics[J]. Journal of Agricultural Biotechnology, 29 (12): 2387-2395.) [4] Basak S, Geng H, Jiang R. J. 2014. Rewiring global regulator cAMP receptor protein (crp) to improve E. coli toler-ance towards low pH[J]. Biotechnology, 173: 68-75. [5] Cai X, Chen H, Blackall J, et al. 2005. Serological character-ization of Haemophilus parasuis isolates from China[J]. Veterinary Microbiology, 111(3-4): 231-236. [6] Chen C, Choudhury A, Zhang S, et al. 2020. Integrating CRIS-PR-enabled trackable genome engineering and transcrip-tomic analysis of global regulators for antibiotic resis-tance selection and identification in Escherichia coli[J]. mSystems, 5(2): e00232-20. [7] Chen Z W, Hsuan S, Liao J W, et al. 2010. Mutations in the Salmonella enterica serovar choleraesuis cAMP-receptor protein gene lead to functional defects in the SPI-1 type Ⅲ secretion system[J]. Veterinary Research, 41(1): 5. [8] Cho Y, Park J, Yu J E. 2017. Edwardsiella piscicida lacking the cyclic AMP receptor protein (crp) is avirulent and immu-nogenic in fish[J]. Fish & Shellfish Immunology, 68:243-250. [9] Geng H, Jiang R. 2015. cAMP receptor protein (crp)-mediat-ed resistance/tolerance in bacteria: Mechanism and utili-zation in biotechnology[J]. Applied Microbiology and Biotechnology, 99(11): 4533-4543. [10] Gwinn M L, Yi D, Smith H O, et al. 1996. Role of the two- component signal transduction and the phosphoenolpyr-uvate: Carbohydrate phosphotransferase systems in com-petence development of Haemophilus influenzae Rd[J].Journal Bacteriology, 178(21): 6366-6368. [11] Huang J, Wang X, Cao Q, et al. 2016. ClpP participates in stress tolerance and negatively regulates biofilm formation in Haemophilus parasuis[J]. Veterinary Microbiolo-gy, 182: 141-149. [12] Jiang C S, Cheng Y F, Cao H, et al. 2020. Effect of cAMP re-ceptor protein gene on growth characteristics and stress resistance of Haemophilus parasuis Serovar 5[J]. Fron-tiers in Cellular and Infection Microbiology, 10:19. [13] Jiang C S, Ren J P, Zhang X Q, et al. 2021. Deletion of thecrpgene affects the virulence and the activation of the NF- κB and MAPK signaling pathways in PK-15 and iPAM cells derived from G. parasuis serovar 5[J]. Veterinary Microbiology, 261: 109198. [14] Lee J Y, Yang K S, Jang S A, et al. 2011. Engineering butanol- tolerance in Escherichia coli with artificial transcription factor libraries[J]. Biotechnology and Bioengineering, 108(4): 742-749. [15] Liu H, Xue Q, Zeng Q, Zhao Z. 2016. Haemophilus parasuis vaccines[J]. Veterinary Immunology and Immunopathol-ogy, 180: 53-58. [16] Ni H B, Gong Q L, Zhao Q, et al. 2020. Prevalence of Hae- mophilus parasuis "Glaesserella parasuis" in pigs in Chi-na: A systematic review and meta-analysis[J]. Preven-tive Veterinary Medicine, 182: 105083. [17] Ou Q, Fan J, Duan D, et al. 2017. Involvement of cAMP re-ceptor protein in biofilm formation, fimbria production, capsular polysaccharide biosynthesis and lethality in mouse of Klebsiella pneumoniae serotype K1 causing pyogenic liver abscess[J]. Journal of Medical Microbiol-ogy, 66(1): 1-7. [18] Yuan X, Cao J, Wang R, et al. 2023. Genetically engineering Escherichia coli to produce xylitol from corncob hydroly-sate without lime detoxification[J]. Molecules, 28(4): 1550. [19] Zahid M S, Awasthi S P, Asakura M, et al. 2015. Suppression of virulence of toxigenic Vibrio cholerae by anethole through the cyclic AMP (cAMP)-cAMP receptor protein signaling system[J]. PLOS ONE, 10: e0137529. [20] Zhang H, Chong H, Ching C B, et al. 2012a. Random muta-genesis of global transcription factor cAMP receptor protein for improved osmotolerance[J]. Biotechnology and Bioengineering, 109(5): 1165-1172. [21] Zhang H, Chong H, Ching C B. 2012b. Engineering global transcription factor cyclic AMP receptor protein of Esch- erichia coli for improved 1-butanol tolerance[J]. Applied Microbiology Biotechnology, 94(4): 1107-1117. [22] Zhao X, Liu Q, Xiao K, et al. 2016. Identification of thecrpgene in avian Pasteurella multocida and evaluation of the effects ofcrpdeletion on its phenotype, virulence and immunogenicity[J]. BMC Microbiology, 16(1): 125. |
|
|
|